The invention relates to a method of and apparatus for determining the rate of enzyme reaction, and more particularly, to such method and apparatus in which the quantity of enzyme in a blood sample is determined by a chemical reaction.
Enzyme represents an organic, high polymer catalyst which is present in biological bodies and which is involved with almost every chemical reaction which takes place within the biological bodies. The quantity of enzyme contained in a blood sample cannot be directly determined and is usually determined by an alternative method in which coenzyme is mixed with a collected blood sample and the absorbance of the coenzyme determined to represent a corresponding quantity of enzyme. Specifically, the quantity of enzyme is derived from the rate per unit time of catalyzed reaction. Such determination of the quantity of active enzyme in a blood sample is commonly referred to as the determination of initial rate of chemical reaction, the determination of activity of enzyme, or the determination of rate of enzyme reaction.
An apparatus used for such determination is arranged to maintain a proportional relationship between the absorbance and the time so that a change thereof can be utilized to determine the enzyme quantity. Specifically, the enzyme quantity IU per liter is determined as follows. EQU IU/l=.DELTA.A/min.times.K (1)
where EQU K=V.sub.t .times.V.sub.f .times.1000/V.sub.s .times.C
and wherein l represents a liter of blood, .DELTA.A/min a difference in the absorbance (optical density) per minute, V.sub.f the overall volume, V.sub.s the volume of sample, V.sub.t a temperature correction coefficient and C a factor which relates to the determination of a coenzyme at a given optical wavelength.
The equation (1) can be rewritten as follows: EQU IU/l=.DELTA.A/min.times.K'.times.V.sub.t ( 2)
where EQU K'=V.sub.f .times.1000/V.sub.s .times.C
Thus it is seen that the enzyme quantity is a function of the temperature.
The difference in the absorbance .DELTA.A can be determined by an optical determination of the initial rate of reaction. Specifically, a reagent is introduced into a reaction solution contained in a reaction vessel, and an initial rate of change per unit time in the absorbance of the reaction solution is considered as representing the degree of reaction. Referring to FIG. 1, where the abscissa represents the reaction time t and the ordinate the optical density (O.D), several curves are shown each of which represent the variation of the density which occurs when the reagent is introduced. The initial rate referred to above is represented by changes .DELTA.E.sub.1, .DELTA.E.sub.2, .DELTA.E.sub.3 in the absorbance per one minute .DELTA.t measured along the initial portion of the linear segment of the curves.
In the apparatus which determines the rate of enzyme reaction, the temperature of the reaction solution is maintained constant by a temperature control of a thermostat so that a fixed value of temperature correction coefficient appearing in the equations (1) and (2) can be used. A first prior art arrangement for this purpose is shown in FIG. 2 where a glass vessel 1 contains a thermostat liquid 2 which is heated by a heater 3. A temperature sensor 4 is introduced into the body of the thermostat liquid to determine the temperature thereof so that the liquid can be maintained at a constant temperature through an automatic control of the heater 3. A cell 5 containing a reaction solution 6 is immersed into the thermostat liquid 2, and an optical flux P of a given optical wavelength is passed therethrough to determine the absorbance.
FIG. 3 shows a second prior art arrangement in which a thermostat block 7 is adapted to be heated by a heating element 8, which is automatically controlled by a temperature sensor 9 in order to maintain the block 7 at a constant temperature. A reaction solution 10 is contained in a cell 11, which is fitted into the block 7. The block 7 is formed with a pair of aligned openings 7a, through which the optical flux P is passed to determine the absorbance.
With the conventional arrangements, it is not possible to achieve a satifactory temperature control of the reaction solution with time or as the ambient temperature changes. It is to be noted that the temperature control of the reaction solution represents an important factor in the determination of the reaction rate, and if the temperature of the reaction solution changes by 1.degree. C., the resulting influence upon the final data will be as much as 10%. It is also to be noted that even if the accuracy of the temperature control is improved as desired, this does not result in improving the accuracy of the temperature control of the reaction solution contained in the cell since the control has been directed to the thermostat itself. Thus, if a reagent is introduced into the reaction solution, there immediately results a reduction in the temperature of the reaction solution. It will thus be seen that the determination has been conducted in the prior art arrangement under the reduced temperature condition.